Prognostic and immunological role of adaptor related protein complex 3 subunit mu2 in colon cancer

The expression levels and prognostic role of AP3M2 in colorectal adenocarcinoma (CRAC) have yet to be fully unveiled. Our study comprehensively investigated the clinical significance of AP3M2 in colorectal cancer through an extensive bioinformatics data mining process (TCGA, GEO, GEPIA, Timer, Ualcan, ROCPLOT, and David), followed by experimental validation. We found AP3M2 is a cancer gene, which can be used to distinguish between colorectal cancer and colorectal adenomas, liver metastasis, lung metastasis, colorectal polyp. Higher AP3M2 expression levels were associated with longer overall survival in colon adenocarcinoma. AP3M2 might be the primary biomarker for oxaliplatin in colon cancer and an acquired resistance biomarker for oxaliplatin and 5-fu. AP3M2 was positively associated with CD274, CTLA4. AP3M2 might be associated with T-cell, NF-kappaB transcription factor activity, and response to hypoxia. AP3M2 could predict chemotherapy effectiveness and prognosis for colon cancer patients. AP3M2 might inhibit tumor growth via influencing tumor-infiltrating immune cells in the context of Tumor microenvironment. AP3M2 plays as an oncogene in CRAC and is suggested as a new potential biotarget for therapy.


The expression of AP3M2 between acquired drug-resistant and parental cell lines
The predictive values of AP3M2 in CRAC Based on the rocplot.plotanalysis, in colon cancer patients, the expression level of AP3M2 was found to be higher in non-responders compared to responders who received oxaliplatin (AUC = 0.693, p-value = 2e−03).However, AP3M2 may not serve as a predictive marker for the effectiveness of 5-fluorouracil (AUC = 0.565, p = 0.081) and Irinotecan (AUC = 0.512, p-value = 0.42).Notably, a high level of AP3M2 might be associated with poor treatment response among colon cancer patients receiving oxaliplatin therapy.Conversely, no significant association between AP3M2 expression and drug response was observed among rectal cancer patients treated

AP3M2 might influence the prognosis of colon cancer via immune regulation
According to the KEGG [7][8][9] and GO enrichment analysis of genes associated with AP3M2 (r > 1 or r < − 1, p-value < 0.05), we identified a potential association between AP3M2 and immune regulation, particularly T-cell immunity.In terms of biological processes, AP3M2 may be involved in T cell activation, lymph node development, positive regulation of NF-kappaB transcription factor activity, and response to hypoxia.Regarding cellular components, AP3M2 may play a role in the T cell receptor complex and alpha-beta T cell receptor complex Α-β.Additionally, the KEGG pathway analysis suggests an association between AP3M2 and the T cell receptor signaling pathway (Fig. 5a).Among the genes related to T-cell immunity (biological process: GO:0042110, T cell activation; cellular component: GO:0042101, T cell receptor complex; GO:0042105, alpha-beta T cell receptor complex; KEGG pathway: hsa04660, T cell receptor signaling pathway), seven genes (CD3E, CD3G, CD247, CD3D, CD8A, CD8B, ZAP70) were found across all these categories.Furthermore,four genes(CD3E,CD3G,CD247,CD8A) showed significant associations with AP3M2 according to Timer database results(Fig.5b).
The results from KEGG and GO enrichment analyses regarding AP3M2(r > 1 or r < − 1, p-value < 0.05) in rectal cancer are presented in Fig, 6.There is no evidence suggesting an association with immune-related functions in rectal cancer.
Moreover, we investigated the relationship between AP3M2 and ABCG2, which was confirmed the energetic association with oxaliplatin resistance.The relationship between AP3M2 and ABCG2 was positive in colon cancer (r = 0.12) and rectal cancer (r = 0.18).(Fig. 11).

The expression of AP3M2 in colon cancer samples and adjacent normal tissues.
Real-time PCR was used to assess the expression of AP3M2 in colon cancer samples and adjacent normal tissues.The level of AP3M2 expression was higher in the tumor group compared to the normal group (p < 0.05) (Fig. 12).

Discussion
Diseases associated with AP3M2 include Farber Lipogranulomatosis and Generalized Epilepsy With Febrile Seizures Plus 10 .Several studies showed that AP3M2 lends credence to a functional role in alcohol preference and withdrawal through GABAergic transmission 4,11,12 .Few studies reveal its role in in tumorigenesis.AP3M2 plays different roles in various cancers, it is an oncogene in Breast invasive carcinoma, Cholangiocarcinoma, Stomach adenocarcinoma, Colon adenocarcinoma, Rectum adenocarcinoma, Esophageal carcinoma, Head and Neck squamous cell carcinoma, Liver hepatocellular carcinoma and Lung squamous cell carcinoma.Meanwhile, it plays an antioncogene role in Glioblastoma multiforme, Kidney Chromophobe and Thyroid carcinoma 13,14 .In our study, enhanced expression of AP3M2 predicts poor prognosis in patients with colon adenocarcinoma, but not rectal adenocarcinoma.Furthermore, AP3M2 might not be some clinic factor such as different stages, age groups, or gender of colorectal cancer.as well as prognostic in rectal cancer.But it is a prognostic biomarker for colon cancer.At the same time, AP3M2 seems can be a biomarker between colon cancer and colonic adenomas, liver metastasis, lung metastasis, colonic polyp.Available databases such as TCGA, GEO do not provide data on gene expression between rectal cancer and other rectal diseases,which prevents "validation" of our findings with clinical samples.
In the last 15 years, a significant progress in the management of CRC was achieved with several new agents licensed (irinotecan, oxaliplatin, capecitabine, bevacizumab, cetuximab and panitumumab) 15,16 .The chemotherapy agents of choice for adjuvant treatment of colorectal cancer with stage higher than T3N0M0 after resection are 5-fluorouracil (5-FU) and oxaliplatin.In the case of metastatic colorectal cancer (mCRC), oxaliplatin and irinotecan are considered as the most significant cytotoxic compounds [17][18][19] .
Resistance to oxaliplatin chemotherapy has become a significant cause for treatment failure and CRC-related death 20 .In our study, AP3M2 was found to be associated with oxaliplatin response in colon cancer.Moreover, we observed an association between AP3M2 and the NF-κB Signaling Pathway with a positive regulation.Studies have revealed NF-κB as one of the stem cell regulatory pathways frequently dysregulated in tumor cells, participating in the regulation of inflammation and leading to chemoresistance against anti-cancer drugs.One of the crucial factors in oxaliplatin resistance is NF-κB via autocrine signaling through IL-1 and IL-8 [21][22][23] .These findings suggest that AP3M2 could indicate oxaliplatin resistance through the NF-κB Signaling Pathway.Besides, overexpression of ABCG2 in oxaliplatin-resistant cells aims to escape from apoptosis induced by Hsi-Hsien Hsu 24 .AP3M2 expression is positively associated with ABCG2 in our study, we hypothesized that AP3M2 might affect the expression of ABCG2 in some way, influencing the acquired drug resistance of Oxaliplatin.However, oxaliplatin seems to induce immunogenic signals on the surface of cancer cells before apoptosis, triggering interferon-gamma production and interaction with toll-like receptor 4 on dendritic cells, resulting in the immunogenic death of cancer cells 25 .This contradicts our conclusion that the overexpression of AP3M2 is positively associated with dendritic cell infiltration in colon adenocarcinoma.
Tumor-infiltrating immune cells (TICs) in the context of tumor microenvironment (TME) are closely associated with clinical outcomes and can regulate tumor development in some way 26 The promotion of tumor cell proliferation, growth and survival could be triggered by cytokines produced by immune cells 27 .Previous studies revealed that M2 macrophages exhibited pro-tumoral effects through interacting with with various cells, including T helper 2 cells, cancer-associated fibroblasts, cancer cells, regulatory T cells (Tregs), plasmacytoid dendritic cells, and myeloid-derived suppressor cells 28 .This further explains that AP3M2 is positively correlated with M2 macrophage levels and myeloid-derived suppressor cells in CRAC in our study.On the other hand, our studies revealed that AP3M2 is related with response to hypoxia, Tumor-tissue hypoxia can convert M1 macrophages to M2 macrophages 29 .Moreover, AP3M2 overexpression in colorectal adenocarcinoma can also increase the amount of immunosuppressive cells, such as regulatory T cells (Treg) and medullary inhibitory cells, to evade immune surveillance, the numbers of these immunosuppressive cells are closely related to a poor prognosis 30,31 .Recent advances in the field 32 of tumor immunology reveals that Tregs are believed to promote tumor development by inefficient activation of the immune system against tumor cells.FOXP3 33,34 , CD72 35,36 , RUNX1 37 , LAG3 38 , CTLA4 39,40 are related to Tregs in the presence of effector cells and contribute to their suppressive activity, suggesting that depletion of Tregs is believed to be a valid therapeutic approach in many cancer types.In The quantity of the immune infiltrate is crucial for mounting an efficient antitumor response.In this regard, the transcriptional level of key genes involved in T cell, and B cell function may reveal deregulation 41 .We identified hub genes (CD3E, CD3G, CD247, CD8A, CD19, CD20(MS4A1),CTLA4,CD279) that were possibly synclastic related to AP3M2 and contribute to the immune response in Colonic Neoplasm.Studies have shown that the expression of CD3G, CD8A, CD19, MS4A1 is related to immune system activation and significantly associated with prognostic factors or disease survival [42][43][44][45][46] .These genes play a possible role in immune-mediated pathways to achieve the elimination of neoplastic cells, affecting the prognosis 47 .On the contrary, CTLA4 (Cytotoxic T lymphocyte antigen 4, CD152), PD1 (programmed cell death 1, CD279) receptors on the surface of activated T cells inhibit the activation of T cells by interacting with their ligands [48][49][50][51] .CD4+ memory activated and resting T cells and Tregs were negatively correlated with CD3E 41 .This supports the idea that AP3M2 affects prognosis through influencing the immune response.More notably, AP3M2 is independent of Immune Regulation in rectal cancer, which explains the different prognosis manifestation in the rectum instead.
Programmed death-ligand 1 (PD-L1, also known as B7-H1 and CD274) is one of the most critical immune checkpoints in CRAC.The binding of programmed cell death protein 1 (PD-1) and its ligand PD-L1 plays a negative regulatory role in the activation of T cells 52,53 .Previous studies have demonstrated that PD-L1 is overexpressed in various cancers including CRC 54,55 .PD-L1 expression is associated with a poor prognosis and inhibits cell proliferation 56 , migration, and invasion in CRC 57 .Besides, PDL1 may regulate immune-independent and intrinsic functions of tumor cells that include tumor cell apoptosis and autophagy 58,59 .Simultaneously, according to the current literature, the expression of PD-L1 is regulated by hypoxia-inducible factor-1 (HIF-1), which could bind to hypoxic response element (HRE) and upregulate the PD-L1 expression, simultaneously cause T-cell apoptosis and function inhibition 60,61 .NF-kB was also important transcription factors upregulating PD-L1 [62][63][64] .NK cell infiltration induces the expression of immune checkpoints such as PD-1 with upregulated Th1 lymphocyte and cytotoxic T cell 65 .In our study, AP3M2 was found to be positively related to PD-L1(CD274), hypoxic response, NF-kB pathway, and NK cell infiltration in colorectal adenocarcinoma.Therefore, we speculate that AP3M2 plays a possible role in colorectal cancer immunotherapy.
Our study comprehensively analyzed the role of AP3M2 in colorectal adenocarcinoma, incorporating data from both TCGA and GEO.Nevertheless, our study has inherent limitations: first, all analyses were based on RNA sequence data.Second, population heterogeneity might exist across different datasets in this study.The absence of in vivo and in vitro experiments to validate our findings should be noted.Further research is warranted to investigate how AP3M2 affects drug effectiveness and colon cancer prognosis.
We hypothesize that AP3M2 functions as an oncogene in CRAC.AP3M2 could potentially predict chemotherapy effectiveness and prognosis for colon cancer patients.Moreover, AP3M2 might inhibit tumor growth by influencing tumor-infiltrating immune cells (TICs) in the context of the tumor microenvironment (TME).It is suggested as a novel potential biotarget for therapy.

Figure 1 .
Figure 1.The relationship between AP3M2 expression and Tumor types: (a) The transcription level of AP3M2 in various cancer types (TIMER database) (p-value < 0.01).(b) The box plot comparing specific AP3M2 expression in normal (left plot) and colon cancer tissues (right plot) was derived from the ualcan database.(c) (p-value < 1E−12).The box plot comparing specific AP3M2 expression in normal (left plot) and rectal cancer tissue (right plot) was derived from ualcan database.(p-value < 7.51E−05).

Figure 2 .
Figure 2. The relationship between AP3M2 expression and Clinical features: (a) The box plot comparing specific AP3M2 expression in different stages was derived from ualcan database in colon cancer.(b) The box plot comparing specific AP3M2 expression in different stages was derived from ualcan database in rectal cancer.(c)The box plot comparing specific AP3M2 expression in various pathologic Lymph node stages was derived from ualcan database in colon cancer.(d) The box plot comparing specific AP3M2 expression in various pathologic Lymph node stage was derived from ualcan database in rectal cancer.(e)The box plot comparing specific AP3M2 expression in different TP53 mutation status was derived from ualcan database in colon cancer.(f) The box plot comparing specific AP3M2 expression in different TP53 mutation status was derived from ualcan database in rectal cancer.

Figure 3 .Figure 4 .
Figure 3.The relationship between AP3M2 expression and survival: (a) The relationship between AP3M2 expression and OS in colon cancer.(b) The relationship between AP3M2 expression and DFS in colon cancer.(c) The relationship between AP3M2 expression and OS in rectal cancer.(d) The relationship between AP3M2 expression and DFS in rectal cancer.

Figure 5 .
Figure 5. Enrichment analysis for AP3M2: (a) The KEGG and GO pathway in colon cancer.(b) The KEGG and GO pathway in rectal cancer.

Figure 6 .Figure 7 .Figure 8 .
Figure 6.The relationship between AP3M2 expression and genes enriched in T-immune: (a) The relationship between AP3M2 expression and CD3E.(b) The relationship between AP3M2 expression and CD3G.(c) The relationship between AP3M2 expression and CD247.(d) The relationship between AP3M2 expression and CD8A.
our study, FOXP3(r = 0.179), CD72(r = 0.155), RUNX1(r = 0.327), LAG3(r = 0.194), CTLA4(r = 0.236) have a positive correlation with AP3M2 in Colon Neoplasm, but no irrelevancy in rectal tumor.Immune check point inhibitors with early Treg depletion might represent an effective strategy for the therapy of Colon Neoplasm 32 , and AP3M2 might be worth considering.This explains the different prognosis of AP3M2 in colon and rectal cancer.

Figure 9 .
Figure 9. Relationship of AP3M2 expression and other genes in colon cancer: (a) The relationship of AP3M2 expression and FOXP3.(b) The relationship of AP3M2 expression and CD72.(c) The relationship of AP3M2 expression and RUNX1.(d) The relationship of AP3M2 expression and LAG3.(e) The relationship of AP3M2 expression and CTLA4.(f) The relationship of AP3M2 expression and PD-L1.(g) The relationship of AP3M2 expression and CD20.(h) The relationship of AP3M2 expression and PD1.(i) The relationship of AP3M2 expression and CD19.

Figure 10 .
Figure 10.Relationship of AP3M2 expression and other genes in rectal cancer: (a) The relationship of AP3M2 expression and FOXP3.(b) The relationship of AP3M2 expression and CD72.(c) The relationship of AP3M2 expression and RUNX1.(d) The relationship of AP3M2 expression and LAG3.(e) The relationship of AP3M2 expression and CTLA4.(f) The relationship of AP3M2 expression and PD-L1.(g) The relationship of AP3M2 expression and CD20.(h) The relationship of AP3M2 expression and PD1.(i) The relationship of AP3M2 expression and CD19.

Figure 11 .
Figure 11.Relationship of AP3M2 expression and ABCG2 in CRAC: (a) the relationship between AP3M2 and ABCG2 in colon cancer.(b) The relationship between AP3M2 and ABCG2 in rectal cancer.

Figure 12 .
Figure 12.The expression of AP3M2 in colon cancer samples and normal tissues.